Targeted therapy has changed cancer treatment, but blocking a single pathway is often ineffective against aggressive solid tumors, due to feedback and activation of alternative pathways. The unique prolyl isomerase Pin1 is abnormally activated in most human tumors and promotes cancer and cancer stem cells by turning on and off at least 42 oncogenes and 21 tumor suppressors, respectively, including many non- druggable transcription factors. Thus, Pin1 is an exciting potential cancer target for simultaneously blocking numerous cancer-driving pathways. However, currently available Pin1 inhibitors lack the specificity and cellular potency required to explore the therapeutic potential of Pin1 as an anti-cancer target. Using the high-affinity Pin1 catalytic peptidomimetic inhibitor pTide as a starting point, we designed and synthesized the first potent covalent Pin1 inhibitors. We have developed efficient biochemical and cellular screening cascade that will guide Pin1 inhibitor optimization. Using these screens, we have identified a number of lead Pin1 inhibitors, including all-trans retinoic acid (ATRA), an approved therapy for acute promyelocytic leukemia (APL), but whose drug target still remains elusive. We discovered that ATRA binds to, inhibits and ultimately induces degradation of active Pin1 selectively in cancer cells. Such ATRA- induced Pin1 ablation exerts potent anticancer activity against APL by degrading PML-RAR? that drives leukemia stem cells, and against triple negative breast cancer (TNBC) by reducing stability of a number of oncogenes and also increasing protein stability of several tumor suppressors. These results provide a rationale for developing more potent and specific Pin1 inhibitors to target a common oncogenic mechanism. In this proposal, we have assembled a multi-disciplinary team that includes Pin1 cancer biology, Pin1 drug screen and inhibitor validation (KP Lu and XZ Zhou, BIDMC), medicinal chemistry (N Gray, DFCI), structural biology (S Dhe-Paganon, DFCI), computational chemistry (N London, Weizmann), and breast cancer translational research (G Wulf, BIDMC). We have discovered an unprecedented means of developing selective Pin1 inhibitors by covalently targeting the critical active site cysteine (Cys113). We have obtained compelling published and preliminary data of the activity of Pin1 inhibitors in a large number of cancers, but here we propose to focus on investigating the potential of targeting Pin1 for the treatment of TNBC, which has the worst prognosis, with no targeted therapy available and where Pin1 plays a critical pathophysiological role. This will be accomplished through a focused medicinal chemistry campaign (Aim 1.1) and guided by detailed mechanistic characterization (Aim 1.2) and co-crystal structural determination (Aim 2), followed by preclinical evaluation in tumor cell and patient-derived xenograft models of TNBC (Aim 3). Our goal is to develop optimized Pin1 inhibitors with properties suitable to interrogate the pharmacological consequences of inhibiting Pin1 enzymatic activity in cellular and in vivo models of TNBC.